When do we speak about galactosaemia?

Galactose Glucose (D)

Lactose

at the root of the intestinal villi ( lactase (β galactosidase ) enzyme

Glucose

( disaccharide )

monosaccharide monosaccharide

monosaccharide

+ H2O

hydrolysis

inhibitid

galactose aldosereductase galactitol

galactokinase

galactose-1-P

gal.-1-P uridyl transferase.

UDP galactose

UDP gal.-4-epimerase

UDP glucose

UDPG pyrophosporilase

glucose-1-P

Total galactose

The process of conversion

cytosol

GLUT

GALT

glucose

galactose

cytosol

The glucose-6-phosphate and the galactose-1-phosphate fall into an ambush in the cell.

THE PHOSPHORYLATION

impermeable

Determination of galactosaemia : based on

Guthrie microbiological metabolic inhibition

method

Principle: the blood-soaked filter paper spots

are placed on agarose medium witch

contains galactose sensitive

mutant E.coli (W5) strain

„Microbiological newborn screening”

Weidemann test: galactose over 20 mg %

Principle: The galactose – dehydrogenase

( Gal-DH ) enzyme transfers H+

from galactose to NAD, to form

fluorescent NADH

Control (mg % ) Sample

20 25 30 35 40

Interfering factors:

False-positive result:

1. if antibiotic was given to the newborn which

inhibits the growth of E-coli

2. any contamination which gives a clear zone

around the disk

False-negative result:

1. if the blood sample was taken before breast

feeding was started

2. if the filter paper was not blood soaked properly

3. if the baby was transfused

Total Galactose Screening Assay/ galactose and galactose-1- phosphate /

R &D Diagnostics Ltd Greece

alkaline phosphatase galactose-1- phosphate Galactose

galactose dehydrogenase

Galactose + NAD+H2O Galaktono (1,4) lakton + NADH++ + H+

diaphorase

NADH + NBT ( yellow ) NBTH ( violet ) + NAD / nitroblu-terazolium / / formazan /

screening plate of galactosaemia

Beutler probe: determination the

deficit of galactose-1-phosphate –galactose-1-phosphate –

uridil- transferase uridil- transferase ( G-1-PUT )

enzyme to confirm the diagnosis of

classical galactosaemia

Samples: heparinised total blood collected into

gas capillary and a healthy control blood

which is collected, transported, stored,

and measured under the same conditions

Principle: In the presence of G-1-PUT the NADP is reduced forming fluorescent NADPH

blood + galactose-1-phosphate + NADP

30‘ 60‘ 90‘ 120‘

Control:

Sample:

Time ( minute ) 0 30 60 90 120

Control

Galaktosaemia .

heterozygote Galaktosaemia classic

Duarte variant

Duarte variant

heterozygote

Detection of Galactose-1-phosphate uridil-transferase /GALT/ enzyme

GALT

GAL-1-P + UDPG Galactose-1-phosphate uridil-transferase G-1-P + UDP-GAL

PGM

G-1-P phosphoglucomutase G-6-P

G-6-PD

G-6-P + NADP glucose-6-phosphate dehydrogenase 6-PGA + NADPH+

6-PGD

6-PGA + NADP 6-fphosphogluconate dehydrogenase R-5-P + NADPH+

ResultsGALT enzyme aktivity :

Normal : > 3,8 U/gHb Transitional: 2,9 -3,8 U/gHb Abnormal: < 2, 9U/gHb

Beteg B.I. T.K. A.G. M.I. M.A F.G. S.A. S.I. T.A.GALT 3.40 3.11 1.05 2.93 3.51 1.77 3.34 1.75 1.25

Galactose tolerance test ( 1,5 g/kg laktóz; max 50g )

30 perc

3,67 3,10 3,12 3,47 3,83

60 perc

4,01 3,33 3,22 3,91 4,11

120 perc

3,81 3,52 3,28 3,88 3,97

Screening year

Screenedsaces

Screening year

Screenedsaces

Screening year

Screenedsaces

1986 1 G1PUT 1996 2 G1PUT5 G-kinase

2006 4 G-kinase

1987 1 G1PUT 1997 2 G1PUT3 G-kinase

2007 4 G-kinase

1988 1 G1PUT 1998 3 G1PUT2 G-kinase2 Duarte?

2008 4 G-kinase

1989 1 G1PUT 1999 2 Duarte?3 G-kinase

2009 4 G-kinase

1990 0 2000 3 G1PUT1 G-kinase

2010 4 G-kinase

1991 1 G1PUT2 G-kinase

2001 02 G-kinase

2011 1 G1PUT

1992 1 G-kinase 2002 2 G1PUT2 G-kinase

1993 1 G1PUT1 G-kinase

2003 1 G1PUT2 G-kinase

1 Duarte?

1994 1 G1PUT1 G-kinase

2004 2 G1PUT0

1995 1 G1PUT1 G-kinase

2005 1 G1PUT 10 G-kinase

Screened galactosaemic cases

Most common point mutations

G-1-PUTransferase Galactokinase

GALT GalK1 GalK2 9p13 17q24 15q21.1

Classical Duarte

Q188R G1391A S142I G171VK285N N314D G148C

P28T

country Screening starting

Austria 1966

Ireland 1972

United Kingdom 1972 Only in Scotland

Switzerland 1975

Hungary 1975

Belgium 1978 Only the Flemish provinces

Germany 1978 Not compulsory in some provinces

Denmark 2002

France no

Netherlands no

Norway no

Poland no

Spain no

Newborn screening of galactosaemia in Europe